1. Field of the Invention
The present invention relates to a method for preparing an Ni-containing magnetic mesoporous silica whose surface strongly binds a histidine-tagged protein, a protein-binding material for degrading a toxic aromatic compound comprising the magnetic mesoporous silica, and a method for degrading a toxic aromatic compound using the magnetic mesoporous silica. More specifically, the present invention relates to a method of preparing a magnetic mesoporous silica responding to a magnetic field by adsorbing a precursor of a transition metal or its ion, such as an iron (Fe) precursor, onto a mesoporous silica, and to a protein-binding material prepared by coating the surface of the magnetic mesoporous silica with transition metal nickel (Ni) or its ion so as to be capable of binding to a specific protein such as histidine-tagged catechol dioxygenase enzyme, and also to a method of degrading a toxic aromatic compound using a histidine-tagged protein immobilized on the magnetic mesoporous silica.
2. Description of the Related Art
Magnetic nanoparticles have received increasing attention in various fields, including separation of biological or chemical substances, cell labeling and sorting, and magnetic resonance imaging, because they are convenient to use, can reduce processing time, and are easy to handle. The use of magnetic nanoparticles for the separation of DNAs and proteomes can reduce time and plays a very important role in increasing efficiency. However, it is not satisfactory for processing large amounts of samples or reducing time. It shows low efficiency because of the agglomeration and low surface reactivity of magnetic nanoparticles.
On the other hand, mesoporous silica materials have controllable pore sizes and large surface areas, and their surface is easy to functionalize. Due to these advantages, mesoporous silica materials have been used in various applications, including catalysts, nanomaterial supports, adsorption and separation processes, and sensors. In the process of preparing such mesoporous silica materials, iron oxide is allowed to react so as to impart magnetic properties to the mesoporous silica materials, such that the mesoporous silica materials can be easily separated from biological mixtures upon the application of an external magnetic field. Due to such properties, the magnetic mesoporous silica materials are expected to be useful for the separation and purification of biomolecules such as proteins or DNAs.
In the prior art, in order to impart magnetic properties to mesoporous silica materials, magnetic nanoparticles such as metals or iron oxides have been imbedded in mesoporous silica materials. However, in this method, magnetic nanoparticles are difficult to distribute uniformly in the aligned pores of mesoporous silica materials, and thus the magnetic nanoparticles clog the pores of the mesoporous silica materials, thereby reducing the surface area and magnetic susceptibility of the mesoporous silica materials. In an attempt to solve this problem, a method of applying magnetic nanoparticles to the wall portion of mesoporous silica materials was proposed, but the kind of magnetic nanoparticles applicable thereto is limited.
Other methods include a hard-templating method comprising depositing magnetic nanoparticles on templates and then removing the templates. The nano-casting method is applied in various ways to make metal oxides which are difficult to make by conventional methods. However, the metal oxides made according to this processing method have a low saturation magnetic field compared to pure metals.
The saturation magnetic field of a mesoporous silica material imparted with magnetic properties is determined by the amount and composition of the magnetic material. The composition of multi-component mesostructured alloys which were recently reported is easier to control than that provided by the hard-templating method. Many approaches have been proposed to develop magnetic mesoporous materials, but there still remain problems to be solved.
A method for the isolation and purification of a protein should have high selectivity for the protein and a minimal effect on the structure of the protein. Among protein purification methods, the use of a tag has good protein selectivity and a minimal effect on the protein structure. Various peptides and proteins are used as tags, and among them, a histidine tag is most frequently used. The histidine tag consists of 6 histidine residues. Thus, the histidine tag is advantageous in that it can purify a protein without influencing the original structure of the protein, because it is small in size. In addition to the histidine tag, a GST (Glutathione S-transferase)-tag is frequently used. The GST tag has protein selectivity much higher than the histidine tag, but it has a shortcoming in that it is large in size so that it should be cleaved after purification of the protein.
An existing method for the isolation and purification of a histidine-tagged protein is carried out using an IMAC column through the reversible binding between transition metal ions (such as Co2+ or Ni2+) and histidine. The IMAC column is prepared by coupling a chelating ligand to the column packing material and then coordinating transition metal ions. However, the packing materials that are used in this method have shortcomings in that the chelating ligand is prepared through a complex organic synthesis process and in that the separation and purification process is time-consuming.
It has long been known that histidine-tagged proteins easily bind to the surface of transition metal oxides. Recently, Professor Hyeon's Group (Seoul National University, Korea) reported the preparation of magnetic nanoparticles using iron oxide and nickel oxide and a technology of effectively separating a histidine-tagged protein using the magnetic nanoparticles (T. Hyeon et al., Nanocomposite Spheres Decorated with NiO Nanoparticles for a Magnetically Recyclable Protein Separation System. Adv. Mater. 2010, 22, 57-60).
As described above, the use of magnetic nanoparticles does not achieve satisfactory separation efficiency due to the agglomeration and low surface reactivity of the magnetic nanoparticles. Also, the existing mesoporous materials have low surface area and saturation magnetic field. In addition, the method of separating a protein using the IMAC column has problems in that a complex organic synthesis process should be carried out to synthesize the chelating ligand and in that a significant amount of time is required to isolate the protein, indicating that the IMAC column is difficult to use in a protein isolation process in which a reaction should be carried out within a short time.